Infiltrating leukocytes confound the detection of E-cadherin promoter methylation in tumors

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Abstract

Promoter hypermethylation is known to result in transcriptional downregulation of many genes including the CDH1 gene. In this study we set out to determine CDH1 promoter methylation in breast tumors with decreased or absent E-cadherin protein expression and without CDH1 gene mutations by methylation-specific PCR (MSP). Interestingly, some tumor samples with normal E-cadherin expression yielded a methylation-specific PCR product. We hypothesized that other cells than tumor cells contribute to these products. Since in normal breast tissue no CDH1 promoter methylation is detected, infiltrating leukocytes, often present in tumors, might account for these methylation-specific fragments. Indeed, a methylation-specific fragment is found in all twelve leukocyte samples tested. Furthermore, activated T-cells also yielded a methylation-specific fragment. Sequencing of these fragments reveals two distinct methylation profiles. Leukocytes have only partial methylation of some CpGs, while the tumor-associated methylation profile shows complete methylation of most CpGs. Therefore, to assess whether CDH1 methylation is tumor associated, sequencing of MSP products is a prerequisite. Here we show that out of six lobular tumors lacking E-cadherin protein expression, three have tumor-associated CDH1 promoter methylation while in three other tumors no methylation is detected.

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Materials and methods

Tumor and normal breast material. Breast tumor tissues were obtained from the Rijnland Hospital (Leiderdorp) and the Leiden University Medical Center. Material was used only with the patient’s consent. The histology of the tumors was reviewed by a pathologist (VTHMBS). Breast tissue from a cosmetic breast reduction surgery sample was obtained from the Rijnland Hospital (Leiderdorp). Samples were snapfrozen in cooled (−70 °C) isopentane for DNA isolation or embedded in paraffin wax for

No correlation between E-cadherin expression and methylation-specific PCR fragments in primary breast tumors

We determined the methylation profile of part of the CDH1 promoter using the MSP method on 19 primary breast tumors: 7 ductal carcinomas, 11 lobular carcinomas, and 1 ducto-lobular carcinoma. E-cadherin protein expression in these tumors has been determined by immunohistochemical staining (Table 1). This set of breast tumors was selected for the absence of CDH1 mutations as detected by single-strand conformation polymorphism and DNA sequencing of the coding sequence and reported previously [10]

Acknowledgements

We thank Dr. J. Calame (Department of Pathology, Rijnland Hospital, Leiderdorp) for providing tissue from a cosmetic breast reduction surgery. Dr. R. van Soest (Department of Hematology, LUMC) is acknowledged for generously providing leukocytes. Activated T-cells were kindly donated by Drs. T.M. Holling and P.J. van den Elsen (Department of Immunohematologie and Blood Transfusion, LUMC). We acknowledge Dr. C.P. Verrijzer (Department of Molecular Cell Biology, LUMC) for useful discussion. Dr.

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